Highly active agonistic CD4 binding site anti-HIV antibodies (HAADS) comprising modified CDRH2 regions that improve contact with gp120

ABSTRACT

Embodiments of the present invention are directed to compositions and methods for anti-HIV (anti-CD4 binding site) potent VRC01-like (PVL) antibodies targeted to gp120 having an amino acid substitution at a residue in the anti-CD4 binding site PVL antibody that is equivalent to Phe43 in CD4, these antibodies having improved potency and breadth.

CROSS-REFERENCE TO RELATED APPLICATION(S)

The present application is a continuation of U.S. patent applicationSer. No. 13/558,312 filed on Jul. 25, 2012, which claims priority to andthe benefit of U.S. Provisional Application Ser. No. 61/511,425 filed onJul. 25, 2011, and U.S. Provisional Application Ser. No. 61/523,244filed on Aug. 12, 2011, the entire contents of both of which areincorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under P01 AI081677-01,RR008862, and RR022220 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

SEQUENCE LISTING STATEMENT

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledCALTE142C1.txt, created Jun. 28, 2019, which is 82,823 bytes in size.The information in the electronic format of the Sequence Listing isincorporated herein by reference in its entirety.

INCORPORATION BY REFERENCE

The present application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy having been amended on Jun.11, 2017, is named “69598SEQLIST.txt” and is 76,303 bytes in size.

TECHNICAL FIELD

This application is directed to a gp120 anti-CD4 binding site(anti-CD4bs) antibody composition that has improved potency and breadthagainst the human immunodeficiency virus, (HIV) which causes acquiredimmunodeficiency syndrome (AIDS).

TECHNICAL BACKGROUND

Three decades after the emergence of HIV there is still no vaccine, andAIDS remains a threat to global public health. However, someHIV-infected individuals eventually develop broadly neutralizingantibodies (bNAbs), i.e., antibodies that neutralize a large panel ofHIV viruses and that can delay viral rebound in HIV patients. Suchantibodies are relevant to vaccine development, as evidenced by theprevention of infection observed after passive transfer to macaques.Antibodies obtained by recent methods target several epitopes on theviral spike gp120 protein. These antibodies show broad and potentactivity, and are referred to as highly active agonistic anti-CD4binding site antibodies (HAADs). HAADS mimic binding of the hostreceptor CD4 protein by exposing the co-receptor binding site on gp120.Despite isolation from different donors, HAADs are derived from twoclosely-related Ig V_(H) genes that share gp120 contact residues (Sheidet al., 2011, Science, 333:1633-1637 and Zhou et al., Science, 2010,329: 811-817.)

Structural analysis of gp120 complexed with VRC01 (a highly potent andbroad HAAD), and gp120 complexed with each of VRC03 and VRC-PG04, (twonew CD4bs antibodies sharing the VRC01 germline V_(H) gene) revealedconvergence of gp120 recognition despite low sequence identities (48-57%in V_(H); 62-65% in V_(L)) (Wu et al, 2011, Science, 333:1593-1602).However, sequence differences between these clonally-unrelated anti-CD4antibodies make it difficult to determine the structural features thatyield neutralization potency and breadth to thereby obtain a potent HIVantibody that is effective across many HIV strains.

SUMMARY

In some embodiments of the present invention, an isolated anti-CD4binding site (anti-CD4bs) potent VRC01-like (PVL) antibody compositionhaving a heavy chain and a light chain includes a substitutedhydrophobic amino acid in the heavy chain at a position equivalent toPhe43 of a CD4 receptor protein. In some embodiments, the positionequivalent to Phe43 of the CD4 receptor protein is position 54 of theheavy chain. In some embodiments, the heavy chain of the anti-CD4bs PVLantibody is selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 45, and/or 46. Insome embodiments, the light chain of the anti-CD4bs PVL antibody isselected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, and/or 43.

In some embodiments of the present invention, the anti-CD4bs PVLantibody is VRC01, VRC02, NIH-45-46, 3BNC60, 3BNC117, 3BNC62, 3BNC95,3BNC176, 12A21, VRC-PG04, VRC-CH30, VRC-CH31, VRC-CH32, VRC-CH33,VRC-CH34, VRC03 heavy chain with VRC01 light chain, gVRC-H5(d74) heavychain with VRC-PG04 light chain, gVRC-H12(d74) heavy chain with VRC-PG04light chain, VRC03, VRC01 heavy chain with VRC03 light chain, 3BNC55,3BNC91, 3BNC104, 3BNC89, 12A21, or VRC-PG04b.

In some embodiments, the substituted hydrophobic amino acid isphenylalanine, tryptophan, or tyrosine.

In some embodiments of the present invention, the anti-CD4bs PVLantibody is NIH45-46, and the heavy chain position equivalent to Phe43of the CD4 receptor protein is 54 of NIH-45-46.

In some embodiments of the present invention, a nucleic acid moleculeencodes for the heavy chain and light chain of an anti-CD4bs PVLantibody having a substituted hydrophobic amino acid in the heavy chainat the position equivalent to Phe43 of the CD4 receptor protein. In someembodiments, a vector includes the nucleic acid molecule encoding theanti-CD4bs PVL antibody. In some embodiments, a cell includes the vectorof the nucleic acid molecule encoding the anti-CD4bs PVL antibody.

In some embodiments of the present invention, a pharmaceuticalcomposition includes the anti-CD4 bs PVL antibody composition or afragment thereof and a pharmaceutically acceptable carrier.

In some embodiments of the present invention, a method of preventing ortreating an HIV infection or an HIV-related disease includes identifyinga patient having an HIV infection or an HIV-related disease, andadministering to a patient a therapeutically effective amount of ananti-CD4bs PVL antibody as described.

In some embodiments of the present invention, a method of increasing thepotency and breadth of an isolated anti-CD4 binding site (anti-CD4bs)potent VRC01-like (PVL) antibody composition having a heavy chain and alight chain includes substituting a target amino acid on the heavy chainwith a substitute hydrophobic amino acid, where the target amino acid isat a position on the heavy chain equivalent to the Phe43 of a CD4receptor protein.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

These and other features and advantages of the present invention will bebetter understood by reference to the following detailed descriptionwhen considered in conjunction with the accompanying drawings.

FIG. 1A is a superimposition of a structural depiction of NIH45-46 Fabalone (in blue) and of NIH45-46-gp120 complex (in magenta), according toembodiments of the present invention.

FIG. 1B is a structural depiction of NIH45-46-gp120 (93TH053) complexand the binding interface and domains labeled and colored as indicated,with NIH45-46 Fab shown in magenta (heavy chain) and light purple (lightchain), and gp120 shown in yellow (inner domain) and grey (outerdomain), according to embodiments of the present invention.

FIG. 2 is a table of the data and refinement statistics from the x-raydiffraction data collected from the NIH45-46 Fab crystal structure andthe NIH45-46-gp120 (93TH057) complex as depicted in FIGS. 1A and 1B,according to embodiments of the present invention.

FIG. 3A is a sequence alignment of the heavy chain variable (V_(H))domains of NIH45-46 (SEQ ID NO: 6) and VRC01 (SEQ ID NO: 2) antibodies,in which the open circles indicate NIH45-46 side chain residues thatcontact gp120 and closed circles indicate NIH45-46 main-chain, ormain-chain and side chain residues that contact gp120, according toembodiments of the present invention.

FIG. 3B is a sequence alignment of the light chain variable (V_(L))domains of NIH45-46 (SEQ ID NO: 5) and VRC01 (SEQ ID NO: 1) antibodies,in which the open circles indicate NIH45-46 side chain residues thatcontact gp120 and closed circles indicate NIH45-46 main-chain, ormain-chain and side chain residues that contact gp120, according toembodiments of the present invention.

FIG. 4A is a superimposition and comparison of a structural depiction ofNIH45-46-gp120 complex (shown in magenta) and a structural depiction ofVRC01-gp120 complex (shown in blue), according to embodiments of thepresent invention.

FIG. 4B is close-up view of the conserved interactions in the gp120contacts of NIH45-46 and VRCO1, with the CD4 binding loop of gp120labeled and shown in yellow, according to embodiments of the presentinvention.

FIG. 5 is a depiction of the interactions of NIH45-46-gp120 complex andVRC01-gp120 complex with NIH45-46 shown in magenta, VRC01 in blue, anddomains of gp120 shown as follows: outer domain (yellow), bridging sheet(orange), CD4 binding loop (blue), V5 loop and D Loop (green), and innerdomain (grey); with the contact region between CDRH3 insertion residuesof NIH45-46 and gp120 shown in the close-up box with insertion residues99a-99b labeled alphabetically, according to embodiments of the presentinvention.

FIG. 6A is a structural depiction of the binding interface of aNIH45-46-gp120 complex characterized by the direct hydrogen bond (dottedline) between the main-chain atom of Gly54_(NIH45-46) (magenta) andAsp368_(gp120) (gray) and two water molecules (larger spheres in dottedline), according to aspects of the present invention.

FIG. 6B is a structural depiction of the contact interface of aCD4-gp120 complex, characterized by CD4 (yellow) forming two directhydrogen bonds (dotted lines) with the CD4-binding loop on gp120,according to embodiments of the present invention.

FIG. 6C is a structural depiction of the contact interface of aVRC03-gp120 complex, characterized by a carbonyl oxygen of Trp54_(VRC03)forming a hydrogen bond with Asp368_(gp120), according to embodiments ofthe present invention.

FIG. 7 is a structural depiction of the binding interface of aNIH45-46-gp120 complex, as shown by a hydrogen bond network between themain-chain carbonyl oxygen of Ala281_(gp120), Tyr99d_(NIH45-46) inCDRH3, and Lys52_(NIH45-46) in CDRH2, in which yellow dots representhydrogen bonds, and as shown in the inset box: a sulfate ion (yellow)substitutes for Ala281_(gp120) in the unbound NIH45-46, according toembodiments of the present invention.

FIG. 8 is a structural depiction of the binding interface of aNIH45-46-gp120 complex, as shown by the electrostatic interactionsbetween Asp99c_(NIH45-46) and Lys97_(gp120) (lower left dotted line) andhydrogen bonds between Asp99c_(NIH45-46)-Tyr97_(NIH45-46) (upper leftdotted line) and Arg99b_(NIH45-46)-Asn99_(gp120) (lower right dottedline), according to embodiments of the present invention.

FIG. 9A is a structural depiction of NIH45-46-gp120 complex (withNIH45-46 shown in magenta and gp120 shown in grey) superimposed with astructural depiction of CD4-gp120 complex (with CD4 shown in yellow andgp120 shown in orange), with an arrow and label of Phe43 of CD4,according to embodiments of the present invention.

FIG. 9B is a close-up view of the superimposition of FIG. 9A with theCDRH2 loop of NIH45-46 (magenta) and the CDR2-like loop of CD4 (yellow)interacting with gp120 (grey surface), according to embodiments of thepresent invention.

FIG. 9C is a structural depiction of a CD4-gp120 (ZM135M.PL10a) complexwith the contact interface labeled and colored as in FIG. 1B, and theinitial site of CD4 attachment is indicated with the oval, according toembodiments of the present invention.

FIG. 9D is a structural depiction of a NIH45-46-gp120 (93TH057) complexwith the contact interface labeled and colored as in FIG. 1B, and thecorresponding Phe43_(CD4) cavity as shown in FIG. 9B is indicated by theasterisk, according to embodiments of the present invention.

FIG. 9E is a structural depiction of a VRC01-gp120 (93TH057) complexwith the contact interface labeled and colored as in FIG. 1B, accordingto embodiments of the present invention.

FIG. 10A is a structural depiction of a NIH45-46-gp120 complexsuperimposed with a VRC01-gp120 complex, in which the Tyr74 showsdifferent interactions with gp120, and the gp120 bridging sheet isdepicted with the broad arrows in gp120 and the asterisks indicate arecombinant Gly₂ linker, according to embodiments of the presentinvention.

FIG. 10B is a close-up view of the structural depiction of FIG. 10Ashowing the hydrogen bond between Tyr74_(NIH45-46) and the main-chaincarbonyl oxygen of Leu122_(gp120), according to embodiments of thepresent invention.

FIG. 11A is a stereo view of a structural depiction of a NIH45-46-gp120complex superimposed with a VRC01-gp120 complex showing thatTyr28_(VRC01 LC) interacts with an N-linked carbohydrate attached toAsn276_(gp120) and the side chain counterpart residue Ser28_(NIH45-46)in the NIH45-46 complex faces away from gp120 to hydrogen bond withArg64_(NIH45-46 LC) (the arrowheads point to Cα atoms of residue 28 ineach structure), according to embodiments of the present invention.

FIG. 11B is a superimposition of NIH45-46LC bound to gp120 (magenta) andunbound (green) showing the hydrogen bonds between Ser28 and Arg64,according to embodiments of the present invention.

FIG. 12 is a structural depiction of gp120 and highlighted differencesin the gp120 resurfaced stabilized core 3 (RSC3) variant, in which theNIH45-46 contact surfaces are shown and the RSC3 mutations shown, withlabeling and coloring as in FIG. 1B, according to embodiments of thepresent invention.

FIG. 13A shows sensorgrams from surface plasmon resonance (SPR)experiments of binding experiments of the 93TH057 gp120 protein withNIH45-46 and NIH45-46^(G54W) Fabs, as indicated, and a table of theK_(D) values is shown, according to embodiments of the presentinvention.

FIG. 13B shows sensorgrams from surface plasmon resonance (SPR)experiments of binding experiments of the CAP244.2.00 D3 gp120 proteinwith NIH45-46 and NIH45-46^(G54W) Fabs, as indicated, and a table of theK_(D) values is shown, according to embodiments of the presentinvention.

FIG. 13C shows sensorgrams from surface plasmon resonance (SPR)experiments of binding experiments of the Q259.d2.17 gp120 protein withNIH45-46 and NIH45-46^(G54W) Fabs, as indicated, and a table of theK_(D) values is shown, according to embodiments of the presentinvention.

FIG. 14 shows neutralization curves for NIH45-46^(G54W) and NIH45-46 instrains DU172.17 and TRO.11, as indicated, according to embodiments ofthe present invention.

FIG. 15A shows a schematic comparing neutralization potencies ofNIH45-46, NIH45-46^(G54W), NIH45-46^(G54F), and NIH45-46^(G54Y), withIC₅₀ values for each color-coded as shown, according embodiments of thepresent invention.

FIG. 15B shows a graphical comparison of neutralization coverage andpotency for VRC01 Monogram (Monogram is a panel of 162 viral strains),VRC01 CAVD (CAVD is a panel of 118 viral strains), PGT121 Monogram,PGT128 Monogram, NIH45-46 CAVD, NIH45-46 hard panel (See Tables 7 and8), and NIH45-46G54W hard panel, according to embodiments of the presentinvention.

FIG. 15C shows neutralization summary spider graphs comparing IC₅₀values for VRC01, NIH45-46, and NIH45-46^(G54W) for 65 common viruses,in which each color represents a different HIV clade, the length of thelines and size of circles are inversely proportional to the IC₅₀ value,the distance between the outer and the inner circle and the distancefrom the inner circle to the center of a spider graph each span twonatural logs in IC₅₀ concentration, the dots on the outer circleindicate strains with IC₅₀ values less than 0.018 μg/ml whose lines weretruncated in the graph, and the size of each dot is inverselyproportional to the IC₅₀ value, according to embodiments of the presentinvention.

FIG. 16 is a schematic illustration of, on the left: PVL antibodyinteractions (magenta and blue-gray) made with gp120 (black); and on theright: CD4 (magenta) with gp120 (black), with the viewpoint of thediagram shown in the inset box, according to embodiments of the presentinvention.

FIG. 17 is a graph of a neutralization assay showing the effects ofmutations at critical residues in YU2 gp120 on neutralization by PVLantibody NIH45-46^(G54W) in which the IC₅₀ values are the mean ofseveral independent experiments, and the graph shows one experiment,according to embodiments of the present invention.

FIG. 18A shows an alignment of the heavy chains of PVL antibodies, theirless potent relatives, and their germ-line precursor (SEQ ID NOs. 2, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 45,46, and 47-59), according to embodiments of the present invention.

FIG. 18B shows an alignment of the light chains of PVL antibodies, theirless potent relatives, and their germ-line precursors (SEQ ID NOs. 1, 5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, and60-73), according to embodiments of the present invention.

DETAILED DESCRIPTION

Aspects of the present invention are directed to anti-CD4 binding site(CD4bs) antibodies. Embodiments of the present invention includeanti-CD4bs antibodies which are potent VRC01-like (PVL) antibodies asdefined herein. In some embodiments of the present invention, ananti-CD4bs PVL antibody has a substituted hydrophobic amino acid residueat a position that is equivalent to phenylalanine at position 43 (Phe43)of the host CD4 receptor protein (CD4). In some embodiments of thepresent invention, a method for increasing the potency and breadth of aPVL antibody includes identifying a target amino acid at the position onthe heavy chain of the PVL antibody that is equivalent to Phe43 on CD4,and substituting the target amino acid with a hydrophobic amino acid.For example, in the PVL antibody, NIH45-46, glycine at position 54(Gly54) is in the Phe43-equivalent position, and substitution of Gly54in NIH45-46 (Gly54_(NIH45-46)) with a hydrophobic amino acid such astryptophan, results in NIH45-46^(G54W) which has increased potency andbreadth compared to NIH45-46.

Abbreviations for amino acids are used throughout this disclosure andfollow the standard nomenclature known in the art. For example, as wouldbe understood by those of ordinary skill in the art, Alanine is Ala orA; Arginine is Arg or R; Asparagine is Asn or N; Aspartic Acid is Asp orD; Cysteine is Cys or C; Glutamic acid is Glu or E; Glutamine is Gln orQ; Glycine is Gly or G; Histidine is His or H; Isoleucine is Ile or I;Leucine is Leu or L; Lysine is Lys or K; Methionine is Met or M;Phenylalanine is Phe or F; Proline is Pro or P; Serine is Ser or S;Threonine is Thr or T; Tryptophan is Trp or W; Tyrosine is Tyr or Y; andValine is Val or V.

Hydrophobic amino acids are well known in the art. Hydrophobic aminoacids include alanine, isoleucine, leucine, methionine, phenylalanine,tryptophan, tyrosine, and valine. In some embodiments of the presentinvention, an anti-CD4bs PVL antibody has a hydrophobic amino acidsubstituted at a position equivalent to Phe43 of the CD4 receptorprotein, wherein the hydrophobic amino acid is alanine, isoleucine,leucine, methionine, phenylalanine, tryptophan, tyrosine, or valine. Inother embodiments, an anti-CD4bs PVL antibody has a hydrophobic aminoacid substituted at the position equivalent to Phe43 of CD4 receptorprotein, wherein the hydrophobic amino acid is tryptophan,phenylalanine, or tyrosine.

Throughout this disclosure and in embodiments of the present invention,the term “antibody” (Ab) as used herein includes monoclonal antibodies,polyclonal antibodies, multispecific antibodies (for example, bispecificantibodies and polyreactive antibodies), and antibody fragments. Thus,the term “antibody” and “isolated antibody” are used interchangeablyherein to refer to an isolated antibody according to embodiments of thepresent invention. An antibody in any context within this specificationis meant to include, but is not be limited to, any specific bindingmember, immunoglobulin class and/or isotype (e.g., IgG1, IgG2, IgG3,IgG4, IgM, IgA, IgD, IgE and IgM); and biologically relevant fragment orspecific binding member thereof, including but not limited to Fab,F(ab′)2, Fv, and scFv (single chain or related entity). It is understoodin the art that an antibody is a glycoprotein comprising at least twoheavy (H) chains and two light (L) chains inter-connected by disulfidebonds, or an antigen binding portion thereof. A heavy chain is comprisedof a heavy chain variable region (VH) and a heavy chain constant region(CH1, CH2 and CH3). A light chain is comprised of a light chain variableregion (VL) and a light chain constant region (CL). The variable regionsof both the heavy and light chains comprise framework regions (FWR) andcomplementarity determining regions (CDR). The four FWR regions arerelatively conserved while CDR regions (CDR1, CDR2 and CDR3) representhypervariable regions and are arranged from the NH2 terminus to the COOHterminus as follows: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, FWR4. Thevariable regions of the heavy and light chains contain a binding domainthat interacts with an antigen while, depending on the isotype, theconstant region(s) may mediate the binding of the immunoglobulin to hosttissues or factors. CDR1, CDR2, and CDR3 of the light chain are referredto as CDRL1, CDRL2 and CDRL3, respectively. CDR1, CDR2, CDR3 of theheavy chain are referred to as CDRH1, CDRH2, and CDRH3, respectively.

Also included in the definition of “antibody” as used herein arechimeric antibodies, humanized antibodies, and recombinant antibodies,human antibodies generated from a transgenic non-human animal, as wellas antibodies selected from libraries using enrichment technologiesavailable to the artisan. The term “variable” refers to the fact thatcertain segments of the variable (V) domains differ extensively insequence among antibodies. The V domain mediates antigen binding anddefines specificity of a particular antibody for its particular antigen.However, the variability is not evenly distributed across the 110-aminoacid span of the variable regions. Instead, the V regions consist ofrelatively invariant stretches called framework regions (FRs) of 15-30amino acids separated by shorter regions of extreme variability called“hypervariable regions” that are each 9-12 amino acids long. Thevariable regions of native heavy and light chains each comprise fourFRs, largely adopting a beta sheet configuration, connected by threehypervariable regions, which form loops connecting, and in some casesforming part of, the beta sheet structure. The hypervariable regions ineach chain are held together in close proximity by the FRs and, with thehypervariable regions from the other chain, contribute to the formationof the antigen-binding site of antibodies. The term “hypervariableregion” as used herein refers to the amino acid residues of an antibodythat are responsible for antigen binding. The hypervariable regiongenerally comprises amino acid residues from a “complementaritydetermining region” (“CDR”).

An antibody of the present invention may be a “humanized antibody”. Ahumanized antibody is considered to be a human antibody that has one ormore amino acid residues introduced into it from a source that isnon-human. These non-human amino acid residues often are referred to as“import” residues, which typically are taken from an “import” variableregion. Humanization may be performed following known methods bysubstituting import hypervariable region sequences for the correspondingsequences of a human antibody. (See, for example, Jones et al., Nature,321:522-525 20 (1986); Reichmann et al., Nature, 332:323-327 (1988);Verhoeyen et al., Science, 239:1534-1536 (1988)) the entire contents ofeach are incorporate herein by reference). Accordingly, such “humanized”antibodies are chimeric antibodies in which substantially less than anintact human variable region has been substituted by the correspondingsequence from a non-human species.

An antibody of the present invention includes an “antibody fragment”which includes a portion of an intact antibody, such as the antigenbinding or variable region of the intact antibody. Examples of antibodyfragments include, but are not limited to, Fab, Fab′, F(ab′)2, and Fvfragments; diabodies; linear antibodies; single-chain antibodymolecules; and multispecific antibodies formed from antibody fragments.(See, for example, U.S. Pat. No. 5,641,870, the entire content of whichis incorporated herein by reference.)

Throughout this disclosure and in embodiments of the present invention,a “potent VRC01-like” (“PVL”) antibody of the present invention is ananti-CD4 binding site antibody that has the following conserved heavychain (HC) and light chain (LC) residues: Arg71_(HC), Trp50_(HC),Asn58_(HC), Trp100B_(HC), Glu96_(LC), Trp67_(LC)/Phe67_(LC), as well asexactly 5 amino acids in CDRL3 domain (using Kabat numbering). (TheKabat numbering system is described in Abhinandan, K. R. and Martin, A.C. R. (2008), “Analysis and improvements to Kabat and structurallycorrect numbering of antibody variable domains,” Molecular Immunology,45: 3832-3839, the entire contents of which are herein incorporated byreference.) A PVL antibody of the present invention is any antibody asdefined herein, that has the listed PVL features irrespective of thesynthesis or derivation of the antibody, irrespective of the otherunrestricted domains of the antibody, and irrespective of whether or notother domains of the antibody are present, so long as the antibody hasthe signature residues and features.

Throughout the disclosure and in embodiments of the present invention,the terms “Phe43-equivalent position” and “Phe43_(CD4) equivalentposition” are used interchangeably and refer to an amino acid positionwithin the heavy chain of a PVL antibody that replicates or mimics thebinding pocket and interface contributed by Phe43 of the host CD4receptor when the CD4 receptor protein is complexed with the HIV viralspike protein gp120. As known in the art, assigned amino acid positionsof an antibody do not necessarily correspond to the amino acid residueas numbered from the amino-terminus. Following the Kabat antibodyresidue/position numbering system, the amino acid residue number may bethe same as the amino acid position, but is not necessarily so. (See,Abhinandan, K. R. and Martin, A. C. R. (2008) Molecular Immunology, 45:3832-3839.) The structure of the antibody peptide determines theposition number. The information for determining position number usingthe Kabat system for each amino acid in a given sequence can bedetermined using the information found in Abhinandan and Martin, 2008.Using this position numbering system, the Phe43-equivalent position in aPVL antibody heavy chain sequence can be determined, and substitutedwith a hydrophobic amino acid to create a similar binding pocket asconferred by Phe43 in CD4. Methods for this mutagenesis are well knownin the art (e.g. Example 2).

Subsequent heavy chain sequences can be analyzed using the Kabatnumbering system to determine the equivalent position to this position54. Alternatively, the Phe43_(CD4)-equivalent position can also bedetermined by structural analysis such as x-ray crystallography. Anymeans of determining the Phe43_(CD4)-equivalent position may be used solong as the Kabat system is followed as applicable.

For example, the Phe43-equivalent position in NIH45-46 is position 54 asdetermined by x-ray crystallography and shown herein. The nativeNIH45-46 sequence contains a glycine at position 54 (Gly54). As such, aPVL antibody substituted with a hydrophobic amino acid at this Phe-43equivalent position mimics the desired contact interface between the CD4receptor protein and the CD4 binding site of gp120 (see, e.g., Example2).

In some embodiments of the present invention, position 54 (Kabatnumbering) of the heavy chain of a PVL antibody has a substitutedhydrophobic amino acid. Position 54 is determined by analyzing a heavychain amino acid sequence of a PVL antibody using the Kabat numberingsystem.

In some embodiments of the present invention, a hydrophobic amino acidis substituted for the “native” amino acid present at thePhe43_(CD4)-equivalent position on the heavy chain of a PVL antibody,where a PVL antibody is an antibody as defined herein having the PVLsignature features as described herein, and “native” refers to the aminoacid that is present in the PVL antibody prior to substitution. Thenative amino acid may also be hydrophobic, and may be substituted withanother hydrophobic amino acid.

In some embodiments of the present invention, non-limiting examples ofPVL antibodies include VRC01, VRC02, NIH-45-46, 3BNC60, 3BNC117, 3BNC62,3BNC95, 3BNC176, 12A21, VRC-PG04, VRC-CH30, VRC-CH31, VRC-CH32,VRC-CH33, VRC-CH34, VRC03 heavy chain (HC) with VRC01 light chain (LC),gVRC-H5(d74)/VRC-PG04LC, and gVRC-H12(d74)/VRC-PG04LC, VRC03, VRC01heavy chain (HC) with VRC03 light chain (LC), 3BNC55, 3BNC91, 3BNC104,3BNC89, 12A21, and VRC-PG04b as listed below in Table 1.

TABLE 1 Examples of PVL Antibodies Light Chain Heavy Chain Antibody NameSEQ ID NO: SEQ ID NO: VRC01 1 2 VRC02 3 4 NIH-45-46 5 6 3BNC60 7 83BNC117 9 10 3BNC62 11 12 3BNC95 13 14 3BNC176 15 16 12A12 17 18VRC-PG04 19 20 VRC-CH30 21 22 VRC-CH31 23 24 VRC-CH32 25 26 VRC-CH33 2728 VRC-CH34 29 30 VRC03 31 32 3BNC55 33 34 3BNC91 35 36 3BNC104 37 383BNC89 39 40 12A21 41 42 VRC-PG04b 43 44 VRC03HC-VRC01LC 1 32VRC01HC/VRC03LC 31 2 gVRC-H5(d74)/ 19 45 VRC-PG04LC gVRC0H12(D74)/ 19 46VRC-PG04LC

In some embodiments of the present invention, a PVL antibody has a heavychain selected from one of the heavy chains listed above in Table 1 (SEQID NOs 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,36, 38, 40, 42, 44, 45, and 46). Any PVL heavy chain may be matched witha PVL light chain so long as the signature PVL residue features aremaintained. In some embodiments, any one of the PVL heavy chains ofTable 1 is expressed with any one of the PVL light chains of SEQ ID NOs1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,39, 41, and 43. In other embodiments, any PVL antibody heavy chain canbe combined with any PVL antibody light chain.

In embodiments of the present invention, the terms “nucleic acid” and“polynucleotide” are used interchangeably herein to refer tosingle-stranded or double-stranded RNA, DNA, or mixed polymers.Polynucleotides can include genomic sequences, extra-genomic and plasmidsequences, and smaller engineered gene segments that express, or can beadapted to express polypeptides.

An “isolated nucleic acid” is a nucleic acid that is substantiallyseparated from other genome DNA sequences as well as proteins orcomplexes such as ribosomes and polymerases, which naturally accompany anative sequence.

In some embodiments of the present invention, nucleic acid moleculesencode part or all of the light and heavy chains of the describedinventive antibodies, and fragments thereof. Due to redundancy of thegenetic code, variants of these sequences will exist that encode thesame amino acid sequences.

The present invention also includes isolated nucleic acid moleculesencoding the polypeptides of the heavy and the light chain of the PVLantibodies listed in Table 1. In some embodiments, an isolated nucleicacid molecule encodes for any of the PVL heavy chain and light chainpolypeptides including those of SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 45, and 46, andSEQ ID NOs 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33, 35, 37, 39, 41, and 43, respectively, in which thePhe43_(CD4)-equivalent amino acid (i.e., the target amino acid) of theheavy chain is substituted with a hydrophobic amino acid.

Embodiments of the present invention also include vectors and host cellsincluding a nucleic acid encoding a PVL antibody of the presentinvention, as well as recombinant techniques for the production ofpolypeptide of the invention. Vectors of the invention include thosecapable of replication in any type of cell or organism, including, forexample, plasmids, phage, cosmids, and mini chromosomes. In someembodiments, vectors comprising a polynucleotide 5 of the describedinvention are vectors suitable for propagation or replication of thepolynucleotide, or vectors suitable for expressing a polypeptide of thedescribed invention. Such vectors are known in the art and commerciallyavailable.

In embodiments of the present invention, “vector” includes shuttle andexpression vectors. Typically, the plasmid construct will include anorigin of replication (for example, the ColE1 origin of replication) anda selectable marker (for example, ampicillin or tetracyclineresistance), for replication and selection, respectively, of theplasmids in bacteria. An “expression vector” refers to a vector thatcontains the necessary control sequences or regulatory elements forexpression of the antibodies including antibody fragment of theinvention, in bacterial or eukaryotic cells.

In some embodiments of the present invention, in order to express apolypeptide of the invention, the nucleotide sequences encoding thepolypeptide, or functional equivalents, may be inserted into anappropriate expression vector, i.e., a vector that contains thenecessary elements for the transcription and translation of the insertedcoding sequence. Methods well known to those skilled in the art may beused to construct expression vectors containing sequences encoding apolypeptide of interest and appropriate transcriptional andtranslational control elements. These methods include in vitrorecombinant DNA techniques, synthetic techniques, and in vivo geneticrecombination. Such techniques are described, for example, in Sambrook,J., et al. (2001) Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Press, Plainview, N.Y., the entire contents of which areincorporated herein by reference.

As used herein, the term “cell” can be any cell, including, but notlimited to, eukaryotic cells, such as, but not limited to, mammaliancells or human cells.

In some embodiments of the present invention, the antibodies disclosedherein are produced recombinantly using vectors and methods available inthe art. (see, e.g. Sambrook et al., 2001, supra). Human antibodies alsocan be generated by in vitro activated B cells (see, for example, U.S.Pat. Nos. 5,567,610 and 5,229,275). Reagents, cloning vectors, and kitsfor genetic manipulation are available from commercial vendors such asBioRad, Stratagene, Invitrogen, ClonTech and Sigma-Aldrich Co.

In some embodiments of the present invention, human antibodies areproduced in transgenic animals (for example, mice) that are capable ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production. For example, it has been describedthat the homozygous deletion of the antibody heavy-chain joining region(JH) gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array into such germline mutant miceresults in the production of human antibodies upon antigen challenge.See, for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551(1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann etal., Year in Immuno., 7:33 (1993); U.S. Pat. Nos. 5,545,806, 5,569,825,5,591,669; 5,545,807; and WO 97/17852, the entire contents of all ofwhich are incorporated herein by reference. Such animals can begenetically engineered to produce human antibodies comprising apolypeptide of a PVL antibody of the present invention.

In some embodiments of the present invention, a method includes thepreparation and administration of an HIV antibody composition (e.g., aPVL antibody having a hydrophobic amino acid substituted at thePhe43_(CD4)-equivalent position of the PVL heavy chain) that is suitablefor administration to a human or non-human primate patient having an HIVinfection, or at risk of HIV infection, in an amount and according to aschedule sufficient to induce a protective immune response against HIV,or reduction of the HIV virus, in a human.

In some embodiments of the present invention, a vaccine includes atleast one antibody as disclosed herein and a pharmaceutically acceptablecarrier. In some embodiments of the present invention, the vaccine is avaccine including at least one PVL antibody as described herein and apharmaceutically acceptable carrier. The vaccine can include a pluralityof the antibodies having the characteristics described herein in anycombination and can further include HIV neutralizing antibodies such asa PVL antibody having the Phe43_(CD4)-equivalent residue on the heavychain substituted with a hydrophobic amino acid.

In some embodiments of the present invention, carriers as used hereininclude pharmaceutically acceptable carriers, excipients or stabilizersthat are nontoxic to the cell or mammal being exposed thereto at thedosages and concentrations employed. Often the physiologicallyacceptable carrier is an aqueous pH buffered solution. Examples ofphysiologically acceptable carriers include, but are not limited to,buffers such as phosphate, citrate, and other organic acids;antioxidants including, but not limited to, ascorbic acid; low molecularweight (less than about 10 residues) polypeptide; proteins, such as, butnot limited to, serum albumin, gelatin, or immunoglobulins; hydrophilicpolymers such as, but not limited to: polyvinylpyrrolidone; amino acidssuch as, but not limited to: glycine, glutamine, asparagine, arginine orlysine; monosaccharides, disaccharides, and other carbohydratesincluding, but not limited to: glucose, mannose, or dextrins; chelatingagents such as, but not limited to: EDTA (ethylenediaminetetraaceticacid); sugar alcohols such as, but not limited to: mannitol or sorbitol;salt-forming counterions such as, but not limited to: sodium; and/ornonionic surfactants such as, but not limited to TWEEN® (polysorbate);polyethylene glycol (PEG), and PLURONICS® (poloxamers).

In some embodiments of the present invention, the compositions mayinclude a single antibody or a combination of antibodies, which can bethe same or different, in order to prophylactically or therapeuticallytreat the progression of various subtypes of HIV infection aftervaccination. Such combinations can be selected according to the desiredimmunity. When an antibody is administered to an animal or a human, itcan be combined with one or more pharmaceutically acceptable carriers,excipients or adjuvants as are known to one of ordinary skilled in theart. The composition can further include broadly neutralizing antibodiesknown in the art, including, for example, a PVL antibody having thePhe43_(CD4)-equivalent residue substituted with a hydrophobic aminoacid.

In some embodiments of the present invention, an antibody-basedpharmaceutical composition includes a therapeutically effective amountof an isolated HIV antibody which provides a prophylactic or therapeutictreatment choice to reduce infection of the HIV virus. Theantibody-based pharmaceutical composition of the present invention maybe formulated by any number of strategies known in the art (e.g., seeMcGoff and Scher, 2000, Solution Formulation of Proteins/Peptides: InMcNally, E. J., ed. Protein Formulation and Delivery. New York, N.Y.:Marcel Dekker; pp. 139-158; Akers and Defilippis, 2000, Peptides andProteins as Parenteral Solutions. In: Pharmaceutical FormulationDevelopment of Peptides and Proteins. Philadelphia, Pa.: Taylor andFrancis; pp. 145-177; Akers, et 5 al., 2002, Pharm. Biotechnol.14:47-127, the entire contents of all of which are incorporated hereinby reference).

In some embodiments of the present invention, a method for treating amammal infected with a virus infection, such as, for example, HIV,comprising administering to said mammal a pharmaceutical compositioncomprising an HIV antibody composition as disclosed herein. According tosome embodiments, the method for treating a mammal infected with HIVincludes administering to said mammal a pharmaceutical composition thatincludes an antibody as disclosed herein, or a fragment thereof. Thecompositions of embodiments of the present invention may include morethan one antibody having the characteristics disclosed herein. Forexample, a plurality or pool of PVL antibodies, each antibody having thePhe43_(CD4)-equivalent residue substituted with a hydrophobic aminoacid.

In some embodiments of the present invention, in vivo treatment of humanand non-human patients includes administering or providing apharmaceutical formulation including an HIV antibody according toembodiments of the present invention. When used for in vivo therapy, theantibodies of the invention are administered to the patient intherapeutically effective amounts (i.e., amounts that eliminate orreduce the patient's viral burden). The antibodies are administered to ahuman patient, in accord with known methods, such as intravenousadministration, for example, as a bolus or by continuous infusion over aperiod of time, by intramuscular, intraperitoneal, intracerobrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation routes. The antibodies can be administeredparenterally, when possible, at the target cell site, or intravenously.In some embodiments, a PVL antibody composition as described herein isadministered by intravenous or subcutaneous administration.

In some embodiments of the present invention, a therapeuticallyeffective amount of an antibody is administered to a patient. In someembodiments, the amount of antibody administered is in the range ofabout 0.1 mg/kg to about 50 mg/kg of patient body weight. Depending onthe type and severity of the infection, about 0.1 mg/kg to about 50mg/kg body weight (for example, about 0.1-15 mg/kg/dose) of antibody isan 5 initial candidate dosage for administration to the patient,whether, for example, by one or more separate administrations, or bycontinuous infusion. The progress of this therapy is readily monitoredby conventional methods and assays and based on criteria known to thephysician or other persons of skill in the art. The above parameters forassessing successful treatment and improvement in the disease arereadily measurable by routine procedures familiar to a physician.

In some embodiments of the present invention, passive immunization usinga PVL antibody as disclosed herein, is used as an effective and safestrategy for the prevention and treatment of HIV disease. (See, forexample, Keller et al., Clin. Microbiol. Rev. 13:602-14 (2000);Casadevall, Nat. Biotechnol. 20:114 (2002); Shibata et al., Nat. Med.5:204-10 (1999); and Igarashi et al., Nat. Med. 5:211-16 (1999), each ofwhich are incorporated herein by reference).

The following Examples are presented for illustrative purposes only, anddo not limit the scope or content of the present application.

EXAMPLES

Reference is made to Diskin et al. 2013, JEM, 210: 1235-1249; Diskin etal., 2011, Science, 334:12989-1293; and West et al., 2012, PNAS, (doi:10.1073/pnas.1208984109), the entire contents of all of which areincorporated herein by reference. FIGS. 18A and 18B show the heavy chain(SEQ ID NOs. 2, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 45, 46, and 47-59) and light chain (SEQ ID NOs. 1, 5, 7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, and60-73) amino acid sequence alignments of several related variant groupsof PVL antibodies as presented in FIG. 2 of West et al. with CDRsdefined using the Chothia definition of the Abysis database.

Example 1. Structural Comparisons of NIH45-46 and VRC01

To determine structural correlates of high potency and breadth in HAADs,structures of NIH45-46 alone and bound to the clade A/E 93TH057 gp120core were solved (FIGS. 1A, 1B and 2). NIH45-46 is a more potent clonalvariant of VRC01 that was isolated from the same donor using a YU2trimer (Sheid et al., 2011, supra), instead of a resurfaced gp120 core(RSC3) as a bait. Comparisons of NIH45-46 Fab in its free versusgp120-bound states demonstrate that gp120 binding does not require majorconformational changes (FIG. 1A). However, gp120 binding induced minorconformational in CDRL1, CDRH3, and in heavy chain framework region 3(FWR3). As predicted by high sequence identity (85% in V_(H); 96% inV_(L)) (FIGS. 3A and 3B), NIH45-46 resembles VRC01 (FIGS. 4A and 4B).However, relative to VRC01, NIH45-46 includes a four-residue insertionwithin CDRH3 (FIG. 5) that was acquired by somatic hypermutation. (See,Sheid et al., 2011, Science, 333:1633-1637, the entire contents of whichare incorporated herein by reference.)

The crystal structure of the NIH45-46-93TH057 gp120 complex verifiedthat NIH45-46 targets the CD4bs on gp120 (FIGS. 1B and 5). The primarybinding surface is the outer domain, including the CD4 binding loop(FIG. 6A), loop D and loop V5, but CDRH3_(NIH45-46) reaches toward thegp120 inner domain (FIG. 1B, 2A-C). Important interactions in theVRC01-93TH057 structure are conserved in NIH45-46 (FIG. 4B); e.g.,residues C-terminal to CDRH2 of VRC01 and NIH45-46 mimic the interactionof main-chain atoms in the C″ β-strand of CD4 domain, which hydrogenbond with the CD4-binding loop of gp120 (FIGS. 6A, 6B, and 6C). In bothNIH45-46 and VRC01, hydrogen bonds between CDRH2 and gp120 arewater-mediated (except for the Gly54_(NIH45-46)/Gly54_(VRC01) carbonyloxygen-Asp368_(gp120) backbone nitrogen H-bond (FIGS. 6A, 6B, and 6C)),and Arg71_(VRC01)/Arg71_(NIH45-46) preserves the Arg59_(CD4) interactionwith Asp368_(gp120). However, the Phe43_(CD4) interaction with ahydrophobic pocket between α-helix 3_(gp120) (CD4 binding loop) andβ-strand 21_(gp120) (bridging sheet) (FIGS. 9A and 9B) is not mimickedby either antibody. Differences between VRC01 and NIH45-46 include theconformation of heavy chain residue Tyr74, a FWR3 residue that wassubstituted during somatic hypermutation (Sheid et al., 2011, supra),and a tyrosine to serine substitution in CDRL1 (FIGS. 10A, 10B, 11A, and11B).

A notable difference between VRC01 and NIH45-46 is the four-residueinsertion (residues 99a-99d) in CDRH3. Three inserted residuescontribute to binding to gp120 (FIG. 5—inset), consistent with deletionof the insertion resulting in about 10-fold reduced neutralizationpotencies (Tables 2 and 3, below).

TABLE 2 In vitro neutralization IC₅₀ values (μg/mL) NIH45-46 NIH45-46NIH45-46 Virus Clade WT Y99dA Δ99a-99d AC10.0.29 B 0.9 4.4 13 TRO.11 B1.9 >50 >50 SC422661.8 B 0.05 0.08 1.4 QH0692.42 B 0.7 2.1 3.7ZM214M.PL15.11 C 0.3 1.1 2.2 CAP45.2.00.G3 C >50 >50 >50 T257-31 CRF020.5 2.4 7.0 (A/G)

TABLE 3 CDRH3 sequence NIH45-46 WT FCTRGKYCTARDYYNWDFEHWGRGAP(SEQ ID NO: 74) NIH45-46 Y99dA FCTRGKYCTARDAYNWDFEHWGRGAP(SEQ ID NO: 75) NIH45-46 Δ99a- FCTRGKYCT----YNWDFEHWGRGAP 99d(SEQ ID NO: 76)

First, the Tyr99d_(NIH45-46) sidechain hydrogen bonds with the loop DAla281_(gp120) carbonyl oxygen (FIG. 7), a main-chain atom, thuspreventing escape through mutation. Indeed, NIH45-46-sensitive strainsaccommodate different sidechains at position 281_(gp120) (Table 4,below).

TABLE 4Comparison of in vitro neutralization for viral strains with differences at 281_(gp120)Residue IC₅₀* Strain gp120 sequence surrounding residue 281 281_(gp120)μg/mL Du156.12 QLLLNGSLAEEEIIIKSENLTDNIKTIIVQLNQSIGINCTRPNNNTRKSV I 0.01((SEQ ID NO: 77) ZM197M.PB7QLLLNGSLAEEEIIIRSENLTDNTKTIIVHLNESVEIECVRPNNNTRKSV T 0.14(SEQ ID NO: 78) ZM214M.PL15QLLLNGSLAEKEIMIRSENLTNNAKTIIVQLTEAVNITCMRPGNNTRRSV A 0.05(SEQ ID NO: 79) ZM249M.PL1QLLLNGSLAEKEIIIRSENITDNVKIIIVHLNESVEINCTRPNNNTRKSI V 0.02(SEQ ID NO: 80) ZM53M.PB12QLLLNGSTAEEDIIIRSENLTNNAKTIIVHLNESIEIECTRPGNNTRKSI A 0.65(SEQ ID NO: 81) ZM109F.PB4QLLLNGSLAEEEIVIRSENLTDNAKTIIVHLNKSVEIECIRPGNNTRKSI A 0.22(SEQ ID NO: 82) ZM135M.PL10aQLLLNGSLSEEGIIIRSKNLTDNTKTIIVHLNESVAIVCTRPNNNTRKSI T 0.36(SEQ ID NO: 83)

The importance of Tyr99d_(NIH45-46) for potency is demonstrated byalanine substitution (NIH45-46 Y99dA), which reduces the neutralizationpotency of NIH45-46 to values intermediate between wild-type NIH45-46and the deletion mutant (Table 2). Second, Asp99c_(NIH45-46) interactselectrostatically with Lys97_(gp120) at the base of α-helix 1_(gp120),and third, Arg99b_(NIH45-46) hydrogen bonds with Asn99_(gp120) (FIG. 8).The conformation of the insertion is stabilized by two intramolecularhydrogen bonds. In one, the Tyr99d_(NIH45-46) sidechain hydrogen bondswith the ε-amino group of Lys52_(NIH45-46) within CDRH2 (FIG. 7), alsoseen in the unbound structure of NIH45-46 (FIG. 7—inset), thus theTyr99d_(NIH45-46) hydroxyl is poised for interacting withAla281_(gp120). A second hydrogen bond between Tyr97_(NIH45-46) andAsp99c_(NIH45-46) in the gp120-bound Fab positions thenegatively-charged aspartic acid for interaction with Lys97_(gp120)(FIG. 8). The region of gp120 with which CDRH3_(NIH45-46) interacts wasnot included in the previously-defined vulnerable site of initial CD4attachment on the gp120 outer domain (FIG. 9C). Thus, gp120 residuesthat contact CDRH3_(NIH45-46) residues required for potentneutralization (Table 2), e.g., Lys97_(gp120), were mutated in RSC3(FIG. 12), the resurfaced gp120 used for isolating bNAbs and as acandidate HIV immunogen.

The insertion in CDRH3 contributes to a higher total buried surface areabetween the NIH45-46 heavy chain and gp120 compared with VRC01 (Table 5,below). The extra contacts with gp120 created by the CDRH3 insertionallow the NIH45-46 footprint on gp120 to more closely resemble the CD4footprint on gp120 than does the VRC01 footprint (FIGS. 9C, 9D, and 9E,and Tables 5A and 5B, below).

TABLE 5A Buried Surface Area (Å²) Interface CDR2 + FWR1 CDR1 FWR2 CDR2FWR3 CDR3 Total Fab on gp120 FWR3₅₆₋₆₅* NIH45-46 HC 0 35 51 181 551 3261144 1097 576 VRC01 HC 0 20 98 136 521 117 892 882 545 NIH45-46 LC 35 80 0 0 159 203 192 0 VRC01 LC 36 114 0 0 0 165 314 367 0 *Residues thatcorrespond to the CDR2 region as defined in Zhou et al., Science, 2010,329: 811-817.

TABLE 5B Inner domain & Outer bridging Loop D + β-15/α-3 + domain exitTotal Interface on sheet NAG NAG V5 β-24 loop gp120 Fab or CD4 NIH45-46328 335 222 292 81 35 1290 1346 VRC01 157 433 208 328 43 57 1225 1206CD4 400 136 263 155 14 97 973 1059

The observation that NIH45-46 shows more extensive contacts relative toVRC01 with the inner domain and bridging sheet of gp120 (FIGS. 9D and9E), yet exhibits higher potency and breadth (Sheid et al., 2001,supra), is inconsistent with the suggestion that increased contact areawith regions outside of the outer domain of gp120 correlate withdecreased neutralization potency and/or breadth (Zhou et al., 2010supra; and Wu et al, 2011, Science, 333:1593-1602). Indeed, the observedCDRH3 contacts with the inner domain imply that thecrystallographically-observed conformation of this region, whetherpre-existing or induced, actively played a role in the affinitymaturation events that resulted in the four-residue insertion withCDRH3.

Example 2. Hydrophobic Amino Acid Substitution at Position 54 ofNIH45-46

Although NIH45-46 increases its contacts with the inner domain/bridgingsheet area of gp120, like VRC01, it lacks a critical CD4 contact to ahydrophobic pocket at the boundary between the gp120 bridging sheet andouter domain made by burying Phe43_(CD4). This residue alone accountsfor 23% of the interatomic contacts between CD4 and gp120, serving as a“linchpin” that welds CD4 to gp120 (Kwong et al., 1998, Nature,393:648-659). On gp120, the Phe43 binding cavity is a binding site ofsmall-molecule CD4 mimics (Madani et al., 2008, Structure,16:1689-1701), and a desirable target for compounds to disrupt CD4-gp120interactions (Kwong et al., 1998, supra), yet it remains unfilled in the93TH057 complexes with VRC01 (Zhou et al., 2010, supra) and NIH45-46. Ina superimposition of a CD4-gp120 structure and NIH45-46-gp120 (FIG. 9B),the Cα atom of heavy chain residue Gly54_(NIH45-46) is only about 1.4 Åfrom the Phe43_(CD4) Cα, suggesting that this important interactionmight be mimicked by substituting Gly54_(NIH45-46) with a largehydrophobic residue. Indeed, residue 54 of VRC03 is a tryptophan, andTrp54_(VRC03) is accommodated within gp120's Phe43 binding cavity tomimic Phe43_(CD4), while still maintaining its main-chain hydrogen bondwith Asp368_(gp120) (PDB 3SE8) (FIGS. 6A-6C). If increasing contactswith the inner domain/bridging sheet enhances antibody activity, assuggested by analysis of the NIH45-46-gp120 structure, then substitutingGly54_(NIH45-46) with a large hydrophobic residue should increase thepotency and breadth of NIH45-46.

A series of NIH45-46 mutants were constructed to test the possibilitythat a hydrophobic sidechain at position 54 in NIH45-46 would improveactivity. First it was verified that substitutions at residue 54 did notinterfere with antigen binding by assessing the ability of one mutant,NIH45-46^(G54W), to bind core gp120s. Surface plasmon resonance (SPR)binding analyses demonstrated that NIH45-46^(G54W) Fab bound core gp120swith slightly higher affinities than did NIH45-46 Fab, with differenceslargely due to slower dissociation rates (FIGS. 13A, 13B, and 13C). Nextmutant IgGs were evaluated in neutralization assays using a panel of sixviruses chosen to include NIH45-46-sensitive and resistant strains(Table 6, below).

TABLE 6 NIH45-46 IC₅₀ (μg/mL) Virus Clade WT G54W G54F G54Y G54I G54MG54L G54H SC422661.8 B 0.06 0.03 0.02 0.06 0.1 0.06 0.1 0.09 AC10.0.29 B0.9 0.2 0.3 0.4 8.6 1.5 1.7 0.6 TRO.11 B 1.0 0.09 0.08 0.1 10 0.3 0.30.2 Du172.17 C >50 0.9 16 >50 >50 >50 >50 >50 CAP210.2.00.E8 C >5041 >50 >50 >50 >50 >50 >50 CAP45.2.00.G3 C >50 6.6 >50 45 >50 >50 >50>50

NIH45-46^(G54W) and NIH45-46^(G54F) showed increased potencies andNIH45-46^(G54W) increased breadth by neutralizing three strains that areresistant to >50 μg/mL of NIH45-46. The apparent increase in breadth islikely due to increased potency as evidenced by the extrapolated IC₅₀for NIH45-46 against strain DU172.17 (FIG. 14).

An additional 82 viruses were tested including 13 NIH45-46-resistant, 14weakly-neutralized, and 55 sensitive strains representing all clades, ofwhich 12 are transmitted founder viruses (FIG. 15A, and Tables 7 and 8,below).

TABLE 7 In vitro neutralization IC₅₀ values (μg/mL) in the “hard panel”of viruses NIH45-46 IC₅₀ (μg/mL) Virus Clade Category WT G54W G54F G54Y6545.v4.c1 AC R >50 18.92 >50 >50 6540.v4.c1 AC R >50 >50 >50 >50CAP45.2.00.G3 C R >50 32.25 >50 >50 Du422.1 C R >50 >50 >50 >50CAP210.2.00.E8 C R >50 >50 >50 >50 3817.v2.c59 CD R >50 >50 >50 >5089-F1_2_25 CD R >50 >50 >50 >50 620345.c01 CRF01_AE R >50 >50 >50 >50T250-4 CRF02_AG R >50 1.33 >50 >50 T278-50 CRF02_AG R >50 >50 >50 >50211-9 CRF02_AG R >50 16.41 >50 >50 3016.v5.c45 D R >50 1.47 5.82 17.89Du172.17 C R >50 3.65 >50 >50 3718.v3.c11 A P 19.61 0.01 0.32 0.30703357.C02 CRF01_AE P 19.17 3.43 7.91 5.60 CNE20 BC P 7.83 0.04 0.530.33 CNE21 BC P 6.01 0.03 0.12 0.07 HIV-16845-2.22 C P 5.00 0.45 0.590.59 C2101.c01 CRF01_AE P 4.24 0.04 0.11 0.09 ZM247v1(Rev-) C P, T/F2.94 0.32 0.28 0.42 ZM233M.PB6 C P 2.50 0.02 0.22 0.16 C1080.c03CRF01_AE P 2.48 0.20 0.36 0.29 THRO4156.18 B P 1.91 0.54 0.89 0.663103.v3.c10 ACD P 1.770 0.200 0.370 0.300 231966.c02 D P 1.640 0.0200.060 0.060 TRO.11 B P 1.610 0.040 0.110 0.080 T251-18 CRF02_AG P 1.3500.260 0.410 0.350 Ce1176_A3 C S, T/F 0.930 0.160 0.240 0.210 QH0692.42 BS 0.720 0.370 0.560 0.520 T255-34 CRF02_AG S 0.710 <0.001 0.030 0.040ZM135M.PL10a C S 0.590 0.040 0.130 0.090 AC10.0.29 B S 0.560 0.130 0.2400.190 T257-31 CRF02_AG S 0.490 0.130 0.170 0.180 CNE58 BC S 0.430 0.0200.040 0.040 Ce0393_C3 C S, T/F 0.334 0.013 0.040 0.036 R1166.c01CRF01_AE S 0.310 0.130 0.400 0.240 CNE30 BC S 0.309 0.052 0.100 0.099CNE17 BC S 0.261 0.036 0.075 0.073 X2131_C1_B5 G S 0.230 0.050 0.1000.100 928-28 CRF02_AG S 0.230 0.060 0.110 0.120 6535.3 B S 0.230 0.0300.070 0.080 ZM53M.PB12 C S 0.175 0.040 0.080 0.060 ZM214M.PL15 C S 0.1700.030 0.090 0.060 Ce703010054_2A2 C S, T/F 0.159 0.027 0.020 0.022ZM197M.PB7 C S 0.150 0.040 0.090 0.070 CAAN5342.A2 B S 0.150 0.070 0.1000.100 Q23.17 A S 0.140 0.010 0.030 0.020 PVO.4 B S 0.120 0.050 0.0700.060 1054_07_TC4_1499 B S, T/F 0.113 0.040 0.076 0.064 Ce2010_F5 C S,T/F 0.101 0.038 0.046 0.049 ZM109F.PB4 C S 0.095 0.002 0.022 0.0261056_10_TA11_1826 B S, T/F 0.094 0.024 0.064 0.044 0330.v4.c3 A S 0.0900.030 0.040 0.030 P1981_C5_3 G S 0.080 0.020 0.030 0.040 Q461.e2 A S0.076 0.009 0.030 0.023 P0402_c2_11 G S 0.073 0.003 0.008 0.012SC422661.8 B S 0.060 0.020 0.040 0.040 62357_14_D3_4589 B S, T/F 0.0600.020 0.040 0.030 WITO4160.33 B S 0.060 0.010 0.020 0.030 Ce2060_G9 C S,T/F 0.058 0.005 0.022 0.021 Ce0682_E4 C S, T/F 0.054 0.010 0.011 0.017231965.c01 D S 0.051 <0.001 0.022 0.025 Q259.d2.17 A S 0.043 <0.0010.009 0.009 TRJO4551.58 B S 0.040 0.010 0.030 0.030 6811.v7.c18 CD S0.035 <0.001 0.017 0.011 R2184.c04 CRF01_AE S 0.034 0.005 0.015 0.0156480.v4.c25 CD S 0.032 0.004 0.014 0.018 X1254_c3 G S 0.032 0.002 0.0110.013 Q842.d12 A S 0.031 0.005 0.011 0.015 C3347.c11 CRF01_AE S 0.029<0.001 0.015 0.011 1006_11_C3_1601 B S, T/F 0.027 <0.001 0.003 0.0053415.v1.c1 A S 0.022 <0.001 <0.001 <0.001 X1193_c1 G S 0.021 <0.001<0.001 0.006 Du156.12 C S 0.020 <0.001 <0.001 0.007 RHPA4259.7 B S 0.017<0.001 0.005 0.007 ZM249M.PL1 C S 0.017 0.002 0.004 0.003 0815.v3.c3 ACDS 0.014 <0.001 <0.001 <0.001 REJO4541.67 B S 0.014 0.002 0.007 0.0073301.v1.c24 AC S 0.009 <0.001 0.001 0.003 Q769.d22 A S 0.009 <0.0010.005 0.007 CNE53 BC S 0.008 <0.001 0.005 0.006 WEAU_d15_410_787 B S,T/F 0.005 <0.001 <0.001 0.002 Geometric means 0.417 0.046 0.120 0.124Category R—Resistant P—Poorly sensitive S—Sensitive T/F—TransmittedFounder

TABLE 8 In vitro neutralization IC₈₀ values (μg/mL) in the “hard panel”of viruses NIH45-46 IC₈₀ (μg/mL) Virus Clade Category WT G54W G54F G54YT250-4 CRF02_AG R >50 44.94 >50 >50 703357.C02 CRF01_AE R >50 17.61 >5030.58 CAP45.2.00.G3 C R >50 >50 >50 >50 CNE20 BC R >50 0.48 3.91 2.40CAP210.2.00.E8 C R >50 >50 >50 >50 T278-50 CRF02_AG R >50 >50 >50 >50211-9 CRF02_AG R >50 >50 >50 >50 620345.c01 CRF01_AE R >50 >50 >50 >503016.v5.c45 D R >50 15.37 36.97 >50 3817.v2.c59 CD R >50 >50 >50 >5089-F1_2_25 CD R >50 >50 >50 >50 6540.v4.c1 AC R >50 >50 >50 >506545.v4.c1 AC R >50 >50 >50 >50 Du422.1 C P >50 >50 >50 >50 3718.v3.c11A P >50 0.04 4.620 3.85 Du172.17 C P >50 42.85 >50 >50 CNE21 BC P 38.070.16 0.66 0.29 C2101.c01 CRF01_AE P 31.37 0.17 0.42 0.27 ZM247v1(Rev-) CP, T/F 24.50 2.60 2.45 3.57 HIV-16845-2.22 C P 22.61 2.10 2.75 2.75ZM233M.PB6 C P 14.18 0.11 0.99 0.78 C1080.c03 CRF01_AE P 11.56 0.91 2.261.83 231966.c02 D P 9.64 0.11 0.24 0.23 THRO4156.18 B P 8.22 1.81 3.012.14 TRO.11 B P 7.49 0.13 0.30 0.22 3103.v3.c10 ACD P 6.15 0.56 1.280.81 T251-18 CRF02_AG P 3.68 0.92 1.38 1.16 T255-34 CRF02_AG S 3.4420.099 0.198 0.174 Ce1176_A3 C S, T/F 3.17 0.45 0.83 0.58 ZM135M.PL10a CS 2.79 0.16 0.43 0.30 CNE58 BC S 2.08 0.05 0.11 0.11 AC10.0.29 B S 1.930.63 1.12 0.90 QH0692.42 B S 1.71 1.12 1.65 1.50 T257-31 CRF02_AG S 1.380.45 0.51 0.67 R1166.c01 CRF01_AE S 1.21 0.51 1.32 0.84 CNE30 BC S 1.0670.196 0.348 0.263 Ce0393_C3 C S, T/F 0.936 0.089 0.173 0.134 X2131_C1_B5G S 0.88 0.24 0.41 0.39 CNE17 BC S 0.734 0.127 0.287 0.264 928-28CRF02_AG S 0.64 0.25 0.41 0.33 ZM53M.PB12 C S 0.61 0.16 0.23 0.22ZM214M.PL15 C S 0.59 0.15 0.30 0.23 ZM197M.PB7 C S 0.55 0.18 0.23 0.216535.3 B S 0.54 0.13 0.27 0.24 Ce703010054_2A2 C S, T/F 0.538 0.0770.070 0.070 Q23.17 A S 0.50 0.03 0.07 0.06 1056_10_TA11_1826 B S, T/F0.447 0.160 0.283 0.189 ZM109F.PB4 C S 0.437 0.070 0.17 0.168 PVO.4 B S0.41 0.16 0.25 0.18 1054_07_TC4_1499 B S, T/F 0.404 0.165 0.283 0.236CAAN5342.A2 B S 0.40 0.21 0.28 0.27 Ce2010_F5 C S, T/F 0.357 0.187 0.1860.235 0330.v4.c3 A S 0.3 0.11 0.13 0.09 Q461.e2 A S 0.291 0.091 0.1350.103 Ce2060_G9 C S, T/F 0.290 0.042 0.085 0.068 WITO4160.33 B S 0.260.04 0.14 0.09 P1981_C5_3 G S 0.24 0.07 0.11 0.09 P0402_c2_11 G S 0.2140.023 0.047 0.049 1006_11_C3_1601 B S, T/F 0.196 0.008 0.024 0.02162357_14_D3_4589 B S, T/F 0.19 0.07 0.14 0.09 Ce0682_E4 C S, T/F 0.1550.039 0.056 0.065 Q259.d2.17 A S 0.154 0.014 0.036 0.034 SC422661.8 B S0.13 0.07 0.10 0.09 TRJO4551.58 B S 0.13 0.05 0.08 0.07 R2184.c04CRF01_AE S 0.127 0.036 0.054 0.045 231965.c01 D S 0.126 0.035 0.0620.054 6811.v7.c18 CD S 0.113 0.033 0.063 0.059 X1254_c3 G S 0.107 0.0180.043 0.041 6480.v4.c25 CD S 0.100 0.021 0.046 0.051 C3347.c11 CRF01_AES 0.094 0.028 0.059 0.052 3415.v1.c1 A S 0.086 0.023 0.029 0.037Q842.d12 A S 0.073 0.025 0.039 0.045 X1193_c1 G S 0.064 0.009 0.0260.024 Du156.12 C S 0.054 0.005 0.019 0.026 ZM249M.PL1 C S 0.053 0.0070.016 0.011 0815.v3.c3 ACD S 0.052 0.003 0.014 0.015 RHPA4259.7 B S0.047 0.007 0.020 0.020 CNE53 BC S 0.039 0.005 0.024 0.027 REJO4541.67 BS 0.035 0.013 0.028 0.020 3301.v1.c24 AC S 0.033 0.004 0.011 0.014Q769.d22 A S 0.033 0.009 0.023 0.024 WEAU_d15_410_787 B S, T/F 0.0150.003 0.004 0.008 Geometric means 1.231 0.225 0.437 0.393 CategoryR—Resistant P—Poorly sensitive S—Sensitive T/F—Transmitted Founder

The above panel of viruses in Tables 7 and 8 (referred to as the “hardpanel”) is more difficult for NIH45-46 to neutralize than arecently-published panel (Sheid et al, 2011, supra) (FIG. 15B).NIH45-46^(G54W) showed increased potency and breadth compared toNIH45-46 and VRC01: geometric mean IC₅₀s of 0.04 μg/mL forNIH45-46^(G54W), 0.41 μg/mL for NIH45-46, and 0.92 μg/mL for VRC01(calculated for 65 viruses against which VRC01 was previously evaluated(Sheid et al, 2011, supra) (Tables 7 and 8, and FIG. 15C). (GeometricIC_(50S) values were calculated without excluding resistant strains byentering values of 50 μg/ml for strains with IC₅₀ values greater than 50μg/ml).

TABLE 9 Sequence correlates of resistance to NIH45-46 Strain 620345_c1Ser456 (Arg) Asp459 (Gly) Lys279 (Asn/Asp) 89_F1_2_25 Ser456 (Arg)Asn458 (Gly) 6540_v4_c1 Ser456 (Arg) Tyr458 (Gly) Ser280 (Asn)6545_v4_c1 Ser456 (Arg) Tyr458 (Gly) Ser280 (Asn) Du422.1 Trp456 (Arg)T250_4 Pro459 (Gly) T278_50 Glu459 (Gly) Ala279 (Asn/Asp) Ce1172_H1deletion of Gly459 X2088_c9 Val459 (Gly) H086.8 Asp459 (Gly)Lys279 (Asn/Asp)

As shown in Table 9, above, 10 of 17 NIH45-46-resistant strains (5 of 7NIH45-46^(G54W)-resistant strains) have amino acid variations atNIH45-46-contacting residues that have a fully conserved residue (shownin parenthesis) in all NIH45-46 sensitive strains. These mutations occurin the β23 strand immediately preceding V5 and in loop D. The positionsof underlined sites have been shown to be important in resistance toVRC01 as reported in Li et al., 2011, J. Virol., 85:8954-8967.

The largest difference between sensitivity to NIH45-46 and sensitivityto VRC01 was in strain 3016.v5.c45 (IC₅₀s of >30 and 0.16 μg/mL,respectively). The most notable residue in 3016.v5.c45 is Tyr282 in loopD. This large residue may alter the conformation of loop D, which isclosely contacted by the four-residue insertion in the NIH45-46 CDRH3.The absence of the insertion may permit VRC01 to better accommodate analtered loop D. The next largest NIH45-46/VRC01 difference, for strainC2101.c1 (12.78 vs. 0.36 μg/mL), may similarly relate to the unusualLys99_(gp120) residue replacing the asparagine that favorably interactswith Arg99b_(NIH45-46) in the NIH45-46-gp120 crystal structure.

From the neutralization assays, it is noted that NIH45-46^(G54W) gainedde novo neutralization activity against six NIH45-46 resistant strains,including the only three that were sensitive to VRC01 but resistant toNIH45-46 in the panel tested in Sheid et al, 2011, supra. For somestrains that NIH45-46 neutralizes poorly, NIH45-46^(G54W) wassignificantly more potent (e.g., improvements of >700-fold for T255-34and 2000-fold for 3718.v3.c11). The enhanced neutralization activity ofNIH45-46^(G54W) implies that Trp54 forms a favorable hydrophobicinteraction with Phe43 cavity of gp120 as seen in VRC03-gp120 (PDB3SE8). NIH45-46^(G54F) showed some increased activity (Tables 6, 7 and8). Substituting Gly54 with tryptophan adds about 140 Å² of buriedsurface area on V_(H) when complexed with gp120, and is consistent withthe reduced dissociation rates observed in surface plasmon resonance(SPR) experiments (FIGS. 13a , 13B, and 13C). By providing a tryptophanin the Phe43 cavity of gp120, NIH45-46^(G54W) may use higher affinitiesand/or slower dissociation rates to overcome incompatible surfacevariations that render some viruses less sensitive or resistant to itseffects.

Heavy chain residue 54 is not conserved in HAADs; in addition to glycine(NIH45-46 and VRC01), residue 54 can be threonine (3BNC60, 3BNC117,3BNC115; VRC-PG04), tyrosine (12A12), phenylalanine (12A21), or arginine(1B2530 and 1NC9), as reported in Sheid et al., 2011, supra; and Wu etal., 2011, supra. Tryptophan substitution in some HAADs was tested andshown in Table 10, below.

TABLE 10 In vitro neutralization IC₅₀ values (μg/mL) 3BNC60 3BNC603BNC117 3BNC117 3BNC55 3BNC55 12A12 12A12 Virus Clade WT T54W WT T54W WTT54W WT Y54W SC422661.8 B 0.1 0.1 0.07 0.07 0.3 0.6 0.2 0.2 AC10.0.29 B13 3.1 6.5 2.8 >50 >50 0.6 0.5 TRO.11 B 0.07 0.06 0.6 0.6 7.6 >50 0.30.2 Du172.17 C 0.05 0.04 0.04 0.9 2 >50 0.2 0.1 CAP210.2.00.E8 C 4.7 5.011 2.8 >50 >50 >50 >50 CAP45.2.00.G3 C 10 19 16 23 >50 >50 0.4 0.2

Passive immunization and/or gene therapy to deliver HIV antibodies isincreasingly being considered as an option for prevention of HIVinfection. To reduce the concentrations and numbers of antibodiesrequired for protection to realistic and affordable levels, highlypotent and broadly neutralizing antibodies are the reagents of choicefor passive delivery. Although it is difficult to compare the potenciesand breadth of antibodies characterized using different virus panels,the natural form of NIH45-46 exhibits superior potency to VRC01 whencompared against a panel of 82 Tier 2 and 3 viruses representing allknown HIV clades (Sheid et al., 2011, supra). One set of HIV antibodies,the PGT antibodies that recognize the gp120 V3 loop and associatedcarbohydrates, exhibited median IC₅₀s up to 10-fold lower than VRC01(Walker et al., 2011, Nature, 477:466-471, the entire contents of whichare incorporated herein by reference), but are less potent and broadthan NIH45-46^(G54W) (FIG. 15B, and Table 11, below).

TABLE 11 IC₅₀ from PGT antibodies and VRC01 using the same virus panelPGT- PGT- PGT- PGT- PGT- PGT- PGT- PGT- PGT- PGT- PGT- Isolate 121 122123 125 126 127 128 130 131 135 136 Include >50 Geometric 0.53 1.03 0.661.85 1.05 2.92 0.39 3.07 7.27 9.53 30.39 (μg/mL) mean Arithmetic 16.6319.39 18.29 25.81 21.75 26.19 15.31 25.39 31.06 34.91 44.02 mean Median0.31 2.02 0.35 34.97 1.08 42.83 0.10 22.98 50.00 50.00 50.00 Exclude >50Geometric 0.07 0.13 0.08 0.09 0.08 0.17 0.06 0.24 0.41 0.32 2.25 (μg/mL)mean Arithmetic 2.17 3.21 2.44 3.34 2.82 2.39 1.56 3.09 2.80 3.88 12.74mean Median 0.03 0.05 0.03 0.04 0.04 0.08 0.02 0.16 0.52 0.17 7.81 %viruses <50 70% 65% 67% 52% 60% 50% 72% 52% 40% 33% 16% (μg/mL) PGT-PGT- PGT- PGT- PGT- PGT- VRC- Isolate 137 141 142 143 144 145 VRC01 PG04PG9 Include >50 Geometric 23.53 3.15 2.40 3.14 13.62 0.91 0.45 0.57 1.27(μg/mL) mean Arithmetic 41.22 24.62 23.30 24.62 33.76 12.83 4.41 7.9215.89 mean Median 50.00 16.01 9.46 13.76 50.00 0.86 0.34 0.30 0.62Exclude >50 Geometric 1.68 0.33 0.24 0.34 1.58 0.30 0.32 0.30 0.36(μg/mL) mean Arithmetic 10.51 3.80 2.99 4.32 6.87 2.59 1.04 1.99 4.33mean Median 3.46 0.35 0.21 0.31 2.06 0.29 0.32 0.20 0.23 % viruses <5022% 55% 57% 56% 38% 78% 92% 88% 75% (μg/mL)

Table 11 above shows a comparison of mean and median IC₅₀ (μg/mL) valuesfor PGT antibodies and VRC01. A direct comparison between NIH45-46 andthe PGT antibodies is not available. However, VRC01 (which was shown ina direct comparison to be less potent than NIH45-46) was directlycompared to the PGT antibodies using the same virus panel. (Sheid etal., 2011, supra.) Mean IC₅₀ values were calculated using data takenfrom Sheid et al., 2011, supra. Geometric and arithmetic means werecalculated to include data for all viral strains (listed as Include >50,in which case, values reported as IC₅₀>50 μg/mL were entered as 50 μg/mLin the calculation) and to exclude viral strains in which the IC₅₀was >50 μg/mL (listed as Exclude >50, in which case the percent of viralstrains with IC₅₀s<50 μg/mL is also reported). Mean IC₅₀s are comparedwith the median IC₅₀s as reported in Sheid et al., 2011, supra.

Contacts between the antibody light chain and gp120 are mostly conservedbetween the NIH45-46-93THO57 and VRC01-93THO57 structures with a notableexception: Ser28_(NIH45-46 LC) in CDRL1 replaces a solvent-exposedtyrosine (Tyr28_(VCR01 LC)) that interacts with ordered N-linkedcarbohydrate attached to Asn276_(93TH057). By contrast, theSer28_(NIH45-46 LC) sidechain does not contact gp120 carbohydrates;instead it faces away from gp120, hydrogen bonding withArg64_(NIH45-46 LC) (FWR3) and creating a 2.7 Å displacement of themain-chain Cα atoms (FIG. 11A). TheSer28_(NIH45-46 LC)-Arg64_(NIH45-46 LC) interaction is maintained inunbound NIH45-46 (FIG. 11B). The position 28 substitution of serine fortyrosine largely accounts for the burial of more surface area in gp120'sinteraction with the VRC01 versus NIH45-46 light chain (681 Å² versus395 Å² total buried surface area; 314 Å² versus 203 Å² buried surfacearea on the light chain) (Tables 5A, 5B). The larger contact area forthe VRC01 light chain may account for the ability of VRC01, but notNIH45-46, to neutralize the clade C CAP45.2.00.G3 strain, given that theNIH45-46 heavy chain paired with the VRC01 light chain neutralizes thisstrain, whereas the VRC01 heavy chain paired with the NIH45-46 lightchain does not (Table 12). However, the VRC01 light chain did notincrease the potency of NIH45-46 against three other viral strains(Table 12), suggesting that the Tyr28 interaction with gp120carbohydrate is not obligatory.

TABLE 12 In vitro neutralization IC₅₀ values (μg/mL) NIH45-46 HC VRC01HC Virus Clade NIH45-46 VRC01 LC NIH45-46 LC VRC01 AC10.0.29 B 0.9 1.04.5 0.8 TRO.11 B 1.9 0.3 24 0.5 SC422661.8 B 0.05 0.2 0.4 0.2 QH0692.42B 0.7 0.9 1.2 0.7 ZM214M.PL15.11 C 0.5 0.6 1.8 0.8 CAP45.2.00.G3 C >502.1 >50 1.8 T257-31 CRF02 0.5 0.6 15 1.0 (A/G)

Example 3. Protein Expression and Purification

Proteins were produced and purified using previously-described methods(Diskin et al., 2010, Nat. Struct. Mol. Biol., 17:608-613, the entirecontents of which are incorporated herein by reference). Briefly,NIH45-46 IgG was expressed by transient transfection in HEK293-6E cells.Secreted IgG was purified from cell supernatants using protein Aaffinity chromatography (GE Healthcare). Fab fragments were prepared bydigesting purified IgG with immobilized papain (Pierce) at 10 mg/mL andthen separating Fabs from Fc-containing proteins using protein Achromatography and Superdex 200 16/60 size exclusion chromatography. Forcrystallization trials, the NIH45-46 Fab for crystallization experimentswas concentrated to 11 mg/mL in 20 mM Tris pH 8.0, 150 mM sodiumchloride, 0.02% sodium azide (TBS). Substitutions in heavy chain residue54 of NIH45-46, 3BNC55, 12A12, 3BNC117 and 3BNC60 were introduced usinga Quikchange II kit (Agilent technologies). Wild type, mutant forms andchain swapped versions of these proteins were expressed as IgGs inHEK293-6E cells and purified by protein A chromatography as describedfor NIH45-46 IgG. Proteins were stored at a concentration of 1 mg/mL forneutralization assays in either 10 mM sodium citrate pH 3.05, 50 mMsodium chloride, 0.02% sodium azide or in TBS (12A12 and 12A12^(Y54W))or in phosphate buffered saline (NIH45-46 mutated/truncated in CDRH3 andNIH45-46/VRC01 heavy and light chain swapped antibodies (Abs)) prior todilution into neutral pH cell media. For SPR analyses, NIH45-46 andNIH45-46^(G54W) heavy chains were subcloned into the pTT5 (NRC-BRI)expression vector to encode C-terminal 6×-His tagged Fab heavy chains(V_(H)—C_(H)1-6×-His tag), and the heavy chain expression vectors wereco-transfected with the appropriate light chain vector into HEK293-6Ecells. Supernatants were collected after 7 days, buffer exchanged intoTBS and loaded on a Ni²⁺-NTA affinity column (Qiagen). Fabs were elutedusing TBS supplemented with 250 mM imidazole and further purified bySuperdex200 10/300 size exclusion chromatography (GE Healthcare) in TBS.

Genes encoding truncated 93TH053, CAP244.2.00.D3, and Q259.d2.17 gp120cores including the deletions and modifications described in Zhou etal., 2010, supra (the entire contents of which are incorporated hereinby reference), were chemically synthesized (BlueHeron). An extradisulfide bond was introduced into 93TH053 by changing the Val65 andSer115 codons into cysteines.

The modified core genes were subcloned into the pACgp67b expressionvector (BD Biosynthesis) to include a C-terminal 6×-His tag, expressedin baculovirus-infected insect cells, and purified from insect cellsupernatants as previously described in Diskin et al., 2010, supra. Forcrystallization experiments, purified NIH45-46 Fab and 93TH057 gp120were incubated at a 1:1 molar ratio and treated with 40 kU ofEndoglycosidase H (New England Biolabs) for 16 hours at 37° C. Thecomplex was purified after the incubation by Superdex 200 10/300 sizeexclusion chromatography (GE Healthcare) and then concentrated toOD₂₈₀=9.6 in 20 mM Tris pH 8.0, 300 mM sodium chloride, 0.02% sodiumazide.

Example 4. Crystallization

Crystallization screening was done by vapor diffusion in sitting dropsby a Mosquito® crystallization robot (TTP labs) using 400 nL drops (1:1protein to reservoir ratio) utilizing commercially availablecrystallization screens (Hampton). Initial crystallization hits for FabNIH45-46 and for NIH45-46-93TH057 complex were identified using thePEGRx HT™ (Hampton) screen and then manually optimized. Thin needle-likecrystals of Fab NIH45-46 (space group P2₁2₁2₁, a=49.4 Å, b=87.4 Å,c=166.4 Å; one molecule per asymmetric unit) were obtained upon mixing aprotein solution at 11 mg/mL with 12% polyethylene glycol 20,000, 0.1 Msodium acetate pH 5.0, 0.1 M sodium/potassium tartrate, 0.02 M ammoniumsulfate at 20° C. Crystals were briefly soaked in mother liquor solutionsupplemented with 15% and then 30% glycerol before flash cooling inliquid nitrogen. Crystals of the NIH45-46-93TH057 complex (space groupP2₁2₁2₁, a=69.1 Å, b=70.5 Å, c=217.7 Å; one molecule per asymmetricunit) were obtained upon mixing a protein solution at OD₂₈₀=9.6 with 12%isopropanol, 10% polyethylene glycol 10,000, 0.1 M sodium citrate pH 5.0at 20° C. Complex crystals were cryo-cooled by covering thecrystallization drops with paraffin oil to prevent evaporation and thenadding an excess of 20% isopropanol, 5% glycerol, 10% polyethyleneglycol, 0.1 M sodium citrate pH 5.0 to the drops prior to mounting andflash cooling the crystals in liquid nitrogen.

Example 5. Data Collection, Structure Solution and Refinement

X-ray diffraction data were collected at the Stanford SynchrotronRadiation Lightsource (SSRL) beamline 12-2 using a Pilatus 6M pixeldetector (Dectris). The data were indexed, integrated and scaled usingXDS as described in Kabsch, 2010, Acta Crystallogr D Biol Crystallogr,66:125-132, the entire contents of which are incorporated herein byreference. The Fab NIH45-46 structure was solved by molecularreplacement using Phaser as described in McCoy et al., 2007, J. Appl.Cryst., 40:658-674, the entire contents of which are incorporated hereinby reference, and the V_(H)V_(L) and C_(H)1 C_(L) domains of the VRC01Fab (PDB code 3NGB) as separate search models. The model was refined to2.6 Å resolution using an iterative approach involving refinement usingthe Phenix crystallography package Adams et al., 2010, Acta CrystallogrD Biol Crystallogr, 66:213-221, the entire contents of which areincorporated herein by reference, and manually fitting models intoelectron density maps using Coot (Emsley et al., 2004, Acta CrystallogrD Biol Crystallogr, 60:2126-2132, the entire contents of which areincorporated herein by reference). The final model (R_(work)=18.4%;R_(free)=23.8%) includes 3380 protein atoms, 125 water molecules and 37ligand atoms, including N-Acetylglucosamine, glycerol and a sulfate ion(FIG. 2). 96.5%, 3.3% and 0.2% of the residues were in the favored,allowed and disallowed regions, respectively, of the Ramachandran plot.The first glutamine of the NIH45-46 heavy chain was modeled as5-pyrrolidone-2-carboxylic acid.

A search model for solving the NIH45-46-93TH057 complex was created bysuperimposing the refined structure of the NIH45-46 Fab on the VRC01 Fabin the structure of VRC01-93TH057 (PDB code 3NGB). A molecularreplacement solution was found as described above using separate searchmodels for the V_(H)V_(L) domains of NIH45-46 complexed with 93TH057 andthe C_(H)1 C_(L) domains of NIH45-46. (FIG. 2). The complex structurewas refined to 2.45 Å resolution as described for the Fab structure. Toreduce model bias, the CDRH3 of NIH45-46 was omitted from the model andthen built into electron density maps after a few rounds of refinement.The final model (R_(work)=20.7%; R_(free)=25.6%) includes 5989 proteinatoms, 67 water molecules and 148 atoms of carbohydrates, citrate andchloride ions (FIG. 2). 96.1%, 3.5% and 0.4% of the residues were in thefavored, allowed and disallowed regions, respectively, of theRamachandran plot. Disordered residues that were not included in themodel were residues 1-2 of the NIH45-46 light chain, residues 133-136and 219-221 of the heavy chain, and residues 302-308 (V3 substitution),residues 397-408 (a total of 6 residues from V4) and the 6×-His tag of93TH057. The first glutamine of the NIH45-46 heavy chain was modeled as5-pyrrolidone-2-carboxylic acid.

Buried surface areas were calculated using AreaIMol in CCP4 and a 1.4 Åprobe. Superimposition calculations were done and molecularrepresentations were generated using PyMol (The PyMOL Molecular GraphicsSystem, Schrödinger, LLC).

Example 6. Surface Plasmon Resonance (SPR) Measurements

The binding of gp120 core proteins to wild-type NIH45-46 Fab and tomutant (NIH45-46^(G54W)) Fab was compared using a Biacore T100instrument (GE Healthcare). Purified NIH45-46 and NIH45-46^(G54W) Fabswere immobilized at coupling densities of 500 resonance units (RU) or1500 RU on a CM5 sensor chip (Biacore) in 10 mM acetate pH 5.0 usingprimary amine coupling chemistry as described in the Biacore manual. Oneof the four flow cells on each sensor chip was mock-coupled using bufferto serve as a blank. Experiments were performed at 25° C. in 20 mMHEPES, pH 7.0, 150 mM sodium chloride and 0.005% (v/v) surfactant P20,and the sensor chips were regenerated using 10 mM glycine, pH 2.5. gp120core proteins were injected in a two-fold dilution series atconcentrations ranging from 500 nM to 31.2 nM at a flow rate of 70μL/min. After subtracting the signal from the mock-coupled flow cell,kinetic data were globally fit to a 1:1 binding model (Biacoreevaluation software) to derive on- and off-rate constants, which wereused to calculate affinities as K_(D)=k_(d)/k_(a).

Example 7. In Vitro Neutralization Assays

A previously-described pseudovirus neutralization assay was used tocompare the neutralization potencies of wild-type and mutant IgGs aspreviously described in Montefiori, 2005, Current protocols inimmunology, Edited by John E. Coligan et al., Chapter 12, Unit 12.11,the entire contents of which are incorporated herein by reference.Briefly, pseudoviruses were generated in HEK293T cells byco-transfection of an Env-expressing vector and areplication-incompetent backbone plasmid. Neutralization was assessed bymeasuring the reduction in luciferase reporter gene expression in thepresence of a potential inhibitor following a single round ofpseudovirus infection in TZM-bl cells. Antibodies were pre-incubatedwith 250 infectious viral units in a three or four-fold dilution seriesfor one hour at 37° C. before adding 10,000 TZM-bl cells per well for atwo-day incubation. Cells were then lysed and luciferase expression wasmeasured using BrightGlo (Promega) and a Victor3 luminometer(PerkinElmer). Nonlinear regression analysis using the program Prism(GraphPad) was used to calculate the concentrations at whichhalf-maximal inhibition was observed (IC₅₀ values) as described in Kleinet al., 2009, PNAS, 106:7385-7390, the entire contents of which areincorporated herein by reference. Samples were initially screened induplicates. Reagents that showed enhanced activity were tested again astriplicates. Values for NIH45-46 and NIH45-46^(G54W) in FIG. 14 wereobtained from three independent experiments. Similar IC₅₀ values wereobtained in two independent neutralization experiments using differentdilution series.

Example 8. Signature Features of PVL Antibodies

The correlation between neutralization potency and the length of two ofthe light chain CDR loops was analyzed in CD4bs antibodies. Therelatively small CDRL1 of VRC01, which has a 2-residue deletion relativeto its germline precursor, was previously correlated with increasedneutralization potency (Zhou et al., 2010, supra). It was noted thatsequences of VRC01, NIH45-46, and VRC-PG04 revealed a more strikingcorrelation for the length of CDRL3, which is only 5 residues in theseantibodies. Examination of the large Abysis database for human Absequences (http://www.bioinf.org.uk/abs/) showed that only about 1% ofV_(L) domains have a CDRL3 length of 5 amino acids, compared with moretypical 9-11 residue lengths. Larger CDRL3 loops would place criticalside chains at the tip of CDRL3 in different locations, thus not able tointeract with gp120 in the same manner. In antibodies with longerCDRL3s, the tip of CDRL3 interacts with Trp47_(HC), a highly conservedresidue (found in 63 of 69 germline V_(H) gene segments) that plays asimilar role as Trp102_(HC) in the Abs with 5-residue CDRL3s tostabilize the V_(H)-V_(L) interface.

V domain alignments revealed the following sequence characteristics ofthe most potent of the VRC01-like Abs: complete conservation of heavychain Arg71_(HC), Trp50_(HC), Asn58_(HC), and Trp102_(HC), and lightchain Glu90_(LC), Trp65_(LC)/Phe65_(LC) and a CDRL3 length of exactly 5amino acids (residues are numbered here as in the structure of NIH45-46;pdb code 3U7Y). Analysis of the per residue variability ofVH1-2*02-derived Abs indicates that the conservation of Trp50_(HC) andAsn58_(HC) is unlikely to be coincidental.

The roles that conserved residues play in the V_(H) domain structure andin binding to the CD4bs on gp120 are shown schematically in FIG. 16 andTable 13, below. The figures are based on interactions present in thegp120 complexes of VRC01, NIH45-46, and VRC-PG04 (Wu et al., 2011,Science, 333:1593-1602; Diskin et al., 2011, Science, 334:1289-1293; andZhou et al., 2010, Science, 329:811-817, the entire contents of all ofwhich are incorporated herein by reference.

TABLE 13 PVL Features PVL Characteristic feature Role Trp50_(HC) H bondwith Asn280_(gp120) Asn58_(HC) H bond with Arg456_(gp120) Arg71_(HC) Hbond/salt bridge with Asp368_(gp120) Trp102*_(HC) H bond withAsn/Asp279_(gp120) Glu90**_(LC) H bond with Gly459_(gp120)Trp65***_(LC)/Phe65***_(LC) interaction with Asn276_(gp120) glycan5-residue CDRL3 prevent steric clashes, position 89_(LC) & 90_(LC) sidechains *Position Trp100B; **Postion Glu96; and ***Trp67/Phe67 usingKabat numbering system.

The side chains of Trp50_(HC), Trp102_(HC), and Trp47_(HC) form anunusual propeller-like arrangement on the surface the V_(H) domain.(Although Trp47_(HC) participates in the “propeller,” it is notconsidered to be a signature residue of potent CD4bs antibodies becauseit is commonly found in V_(H) domains.) The main interactions of thecharacteristic V_(H) domain residues with gp120 are as follows:Trp50_(HC): indole N—H hydrogen bonds with the side chain oxygen ofAsn280_(gp120); Asn58_(HC): side chain N—H hydrogen bonds with thebackbone carbonyl of Arg456_(gp120); Arg71_(HC): side chain hydrogenbonds/salt bridges with the side chain of Asp368_(gp120); andTrp102_(HC): indole N—H hydrogen bonds with the side chain oxygen ofAsn/Asp279_(gp120). Trp102_(HC) also buries 85 Å² of surface area at theV_(H)/V_(L) interface—contacting residues Tyr89_(LC) and Glu90_(LC).

In the light chains, the side chain of Glu90_(LC) forms a hydrogen bondwith the backbone nitrogen of Gly459_(gp120) and/or the side chain ofAsn280_(gp120). The conservation of Trp65_(LC)/Phe65_(LC) is surprisingas this position is distant from gp120 in the available crystalstructures.

For those interactions that depend on specific gp120 side chains, thedegree of conservation of the relevant gp120 residues is 96.4% forAsn/Asp279_(gp120), 96.4% for Asn280_(gp120), and 99.7% forAsp368_(gp120) (based on the 2010 filtered web alignment of 2869 HIV-1sequences in the Los Alamos HIV database; http://www.hiv.lanl.gov/).Arg456_(gp120), which is involved in a main-chain hydrogen bond with thesidechain of Asn58_(HC), is also highly conserved (95.0%).

An SPR-based binding assay demonstrated detectable binding of thegermline heavy chain/mature light chain IgG to immobilized gp140trimers. Binding of germline heavy chain IgGs was analyzed withsubstitutions in the four signature heavy chain residues (W50S, N58S,R71T, and W102S) (again paired with the mature 3BNC60 light chain). TheW50S, R71T, and W102S mutants showed little or no gp140 binding, and theN58S mutation diminished binding by about 20-fold, consistent with thecorresponding PVL characteristic residues playing key roles inrecognition of the HIV-1 envelope spike by the germline PVL B cellreceptor.

To examine the importance of the signature PVL residues to theiractivity, the gp120 sequences of HIV-1 strains resistant toneutralization by NIH45-46 were analyzed. The gp120 residue variantsassociated with resistance were identified by three criteria: first,they are contact residues with NIH45-46; second, they are absent inNIH45-46-resistant viruses; third, they do not appear inNIH45-46-sensitive viruses. The critical positions identified were279_(gp120), 280_(gp120), 456_(gp120), 458_(gp120), and 459_(gp120); thecommon (i.e., sensitive) residues at these positions are Asx, Asn, Arg,Gly, and Gly, respectively, where Asx is Asp or Asn. These sites makesignificant contacts with the characteristic PVL residues (FIG. 16).Viral stains with variations at these sites are generally neutralizedpoorly by all PVL antibodies, as expected if substitutions at thesepositions interfere with common interactions.

To verify the significance of gp120 variations at these positions, pointmutants within the gp160 gene of HIV-1 strain YU2 were engineered,created pseudoviruses carrying the mutant gp160s, and determined theneutralization potencies of the PVL NIH45-46^(G54W) (Diskin et al.,2011, supra) (as characterized by IC₅₀ values). Mutations at 279_(gp120)and 280_(gp120) rendered the virus resistant to neutralization byNIH45-46^(G54W), and substitution of 458_(gp120) diminished theneutralization potency by >1500-fold (FIG. 17).

As disclosed throughout and evidenced by, for example, theneutralization assays of FIG. 14, a PVL antibody such as NIH45-46,having a hydrophobic amino acid (Trp) substituted at thePhe43_(CD4)-equivalent residue (position 54) has increased potency andbreadth in HIV strains. Furthermore, methods are provided foridentifying a PVL antibody and the Phe43_(CD4)-equivalent residue forsubstitution with a hydrophobic amino acid.

While the present invention has been illustrated and described withreference to certain exemplary embodiments, those of ordinary skill inthe art will understand that various modifications and changes may bemade to the described embodiments without departing from the spirit andscope of the present invention, as defined in the following claims.

What is claimed is:
 1. A composition comprising a human anti-CD4 bindingsite (anti-CD4bs) VRC01-related antibody variant having an isolatedlight chain and isolated heavy chain, the heavy chain comprising anamino acid (X) substitution at position 54 selected from G54X, T54X, orS54X according to Kabat numbering, the amino acid (X) being selectedfrom the group consisting of alanine, isoleucine, leucine, methionine,phenylalanine, tryptophan, tyrosine, valine, histidine, arginine,glutamine, asparagine, lysine, glutamic acid, and aspartic acid, theanti-CD4bs VRC01-related antibody variant comprising: a complementaritydetermining region (CDR) 1 of the heavy chain (CDRH1) having a sequenceof Gly26-Tyr27-Glu28-Phe29-(Ile/Leu)30-(Asn/Asp)31-Cys32; (SEQ ID NO:84) a CDR 2 of the heavy chain (CDRH2) having a sequence ofLys52-Pro52A-Arg53-Gly54-Gly55-Ala56 (SEQ ID NO: 85); a CDR 3 of theheavy chain (CDRH3) having a sequence selected from:Gly95-Lys96-(Asn/Tyr)97-Cys98-(Asp/Thr)99-Tyr100-Asn100A-Trp100B-Asp100C-Phe100D-Glu101-His102(SEQ ID NO: 86) orGly95-Lys96-(Asn/Tyr)97-Cys98-(Asp/Thr)99-Ala100-Arg100A-Asp100B-Tyr100C-Tyr100D-Asn100E-Trp100F-Asp100G-Phe100H-Glu101-His102(SEQ ID NO: 87); a CDR 1 of the light chain (CDRL1) having a sequence ofArg24-Thr25-Ser26-Gln27-(Ser/Tyr)28-Gly29-Ser30-Leu33-Ala34 (SEQ IDNO:88); a CDR 2 of the light chain (CDRL2) having a sequence ofSer50-Gly51-Ser52-Thr53-Arg54-Ala55-Ala56; (SEQ ID NO:89) and a CDR 3 ofthe light chain (CDRL3) having a sequence ofGln89-Gln90-Tyr91-Glu96-Phe97 (SEQ ID NO:90), wherein the CDRs aredefined according to the Chothia definition.
 2. The composition of claim1, wherein the heavy chain substitution is tryptophan, tyrosine,phenylalanine, histidine, arginine, glutamine, or asparagine.
 3. Thecomposition of claim 1, wherein the human anti-CD4bs antibody is capableof binding to gp120 at positions corresponding to 279, 280, 368, 458,and 459 according to pdb code 3U7Y.
 4. A pharmaceutical compositioncomprising the human anti-CD4bs antibody of claim 1 or a fragmentthereof, and a pharmaceutically acceptable carrier.
 5. A method ofinhibiting an HIV infection or an HIV-related disease, comprising:administering a therapeutically effective amount of the composition ofclaim 1 to a patient.